Recombinant gp90 protein expressed in Pichia pastoris induces a protective immune response against reticuloendotheliosis virus in chickens.

Reticuloendotheliosis virus (REV) causes an oncogenic, immunosuppressive and runting syndrome in multiple avian hosts worldwide. In this study, the gp90 protein of REV was secretory expressed in Pichia pastoris with high production level and good antigenicity. To fully utilize the expression potential of the P. pastoris expression system, a panel of Pichia clones carrying increasing copies of the gp90 expression cassette was created using an in vitro multimerization approach and the effects of gene dosage on gp90 expression were investigated. Results demonstrated that an increase in gp90 copy number can significantly improve the yields of gp90 protein. Following expression and scale-up, the gp90 protein production level could reach up to 400mg/L, and the protein could be detected by gp90-specific monoclonal antibody. Investigations of its vaccine efficacy demonstrated that the recombinant gp90 protein was able to induce sustained high levels of antibodies against REV as being detected by ELISA and virus neutralizing test. Furthermore, immunization of chickens with the recombinant gp90 vaccine fully protected the animals from viremia after REV infection. Overall, the yeast-expressed gp90 protein retains good immunogenicity and could be used as a potential subunit vaccine candidate for REV prevention.

Sequence analysis for the complete proviral genome of reticuloendotheliosis virus Chinese strain HA9901.

The genomic DNA extracted from chicken embryo fibroblast (CEF) infected with a Chinese field isolate HA9901 of reticuloendotheliosis virus (REV) was used as the template to amplify the REV proviral genomic cDNA by PCR with 6 pairs of primers according to published sequences. Six overlapping fragments were amplified, cloned into the TA vector and sequenced, including a fragment which was amplified from the circular proviral cDNA and covering both 5'- and 3'-ends. The complete sequence of the whole genome was established and analyzed with a DNAstar software. Comparisons of the sequence with two other strains demonstrated that the genomes of REV were relatively conservative, the homogenecity for all genes or LTR fragments of the 3 strains was over 92%, no matter whether they were isolated from different species and regions in different years. But, the homology of Chinese strain HA9901 to a fowl pox virus-associated strain from Chickens was higher than that to strain SNV isolated from ducks.

Alteration of location of dimer linkage sequence in retroviral RNA: little effect on replication or homologous recombination.

Retrovirus particles contain a dimer of retroviral genomic RNA. A defined region of the retrovirus genome has previously been shown to be important for both dimerization and encapsidation. To study the importance of the position of this encapsidation and dimerization signal for retroviral replication and homologous recombination, we used a previously described spleen necrosis virus-based helper cell system. This system allows retroviral vectors with multiple genetic markers to be studied after a single cycle of retroviral replication. The sequence responsible for dimerization, the encapsidation/dimer linkage sequence (E/DLS), was moved from its normal location near the 5' end of the retroviral genome to a location near the 3' end of the genome. We characterized four pairs of retroviral vectors: (i) with both E/DLSs at the 5' ends of the genomes, (ii) with both E/DLSs at the 3' ends of the genomes, and (iii) two with one E/DLS at the 5' end of the genome and one at the 3' end of the genome. We found that moving the E/DLS to the 3' end of the genome resulted in at most an approximately factor of 5 reduction in virus titer in a single cycle of retroviral replication. Furthermore, we found no changes that were attributable to the alteration of the position of the E/DLS in the minus-strand DNA primer transfers or the plus-strand DNA primer transfers, the rate of homologous recombination, or the number of internal template switches in recombinant proviruses. These results indicate that any alignment or conformation necessary for retroviral replication or recombination is not the result of the position of the E/DLS.

Characterization and comparison of avian and murine helper cell lines for production of replication-defective retroviruses for avian transformation.

Several approaches were taken to identify improved helper cell lines for the production of replication-defective avian retroviral vectors for avian transformation. Both QT6 and D17 cells were engineered to become helper cell lines for the production of reticuloendotheliosis virus vectors. The results showed that the majority of lines from the D17, QT6, and D17C3 cells produced titers in the 10(2) to 10(3) cfu/mL range, with one QT6 line producing 10(5) cfu/mL. This high producer line was relatively free of helper virus when restricted to low passage. An amphotropic murine cell line produced a 6- to 10-fold higher amount of virus and had a comparable higher titer on chicken cells, suggesting possible application to avian transformation.

Transactivation of gene expression by nuclear and cytoplasmic rel proteins.

Transcriptional activation of gene expression by oncogenic proteins can lead to cellular transformation. It has recently been demonstrated that the protein encoded by the v-rel oncogene from reticuloendotheliosis virus strain T can transactivate gene expression from certain promoters in a cell-specific manner. We have examined the cytological location, transforming properties, and transactivation properties of proteins encoded by chimeric turkey v-rel/chicken c-rel genes. We found that whereas the v-rel protein was nuclear in both chicken embryo and rat fibroblasts, the presence of the C terminus of the c-rel protein inhibited nuclear localization of the rel protein in these fibroblasts. Cytoplasmic rel proteins containing C-terminal c-rel sequences transactivated gene expression from the polyomavirus late promoter as efficiently as did similar rel proteins located in the nucleus. These results indicate that the cellular location of rel proteins is not important for transactivation of gene expression and suggest that transactivation by rel proteins is indirect, perhaps by affecting an intracellular signal transduction pathway that eventually results in the alteration of gene expression. The transforming properties of the rel protein were unaltered by the presence of the c-rel C terminus, but, as previously reported for turkey c-rel sequences, substitution of chicken c-rel sequences for internal v-rel sequences reduced the transforming activity of the rel protein and eliminated the immortalization ability. However, all of the chimeric v/c-rel proteins were able to transactivate gene expression, indicating that transactivation does not correlate with transformation. These results suggest that transactivation may be necessary but is not sufficient for transformation by rel proteins.

Insertional activation of c-myc by reticuloendotheliosis virus in chicken B lymphoma: nonrandom distribution and orientation of the proviruses.

Chicken syncytial virus, a member of the reticuloendotheliosis virus family, can induce chicken B lymphomas indistinguishable from those caused by avian leukosis virus. Previously, we have demonstrated that the chicken syncytial virus proviruses in these tumors are linked to the proto-oncogene c-myc. We have now determined the arrangement of chicken syncytial virus proviruses in 22 tumors. The results indicate that these proviruses, without exception, are integrated upstream from the second c-myc exon. At least 70% of these insertion sites are clustered in a 0.5-kilobase region immediately preceding the exon. The proviruses are all arranged in the same transcriptional orientation as c-myc. This type of provirus organization bears strong resemblance to that of the avian leukosis virus proviruses involved in c-myc activation.

Pathogenesis of reticuloendotheliosis virus infection in ducks.

A new isolate of reticuloendotheliosis virus (REV) from ducks, RU-1, was used to experimentally infect white pekin ducks. Embryonal or neonatal infection usually resulted in persistent viremias, no REV antibody development, and inability to mount antibody responses against bovine serum albumin (BSA) or sheep red blood cells (SRBC). In contrast, infection at 21 days of age resulted in transient viremias, which terminated coincident with REV antibody development. These ducks remained persistently infected, however, based on virus isolations from peripheral blood lymphocytes. Ducks infected at that age were immunologically competent against BSA and SRBC unless they had been embryonally bursectomized, in which case they behaved virologically and immunologically like those infected at an early age. Bursectomy by itself did not prevent responses against the antigens. Total mortality during 4- or 6-month experimental periods ranged from 80 to 100% in REV-infected groups, regardless of age at infection, route of exposure, or whether the ducks were intact or bursectomized. Most deaths were from non-neoplastic conditions (stunting, bacterial infections), but 17 of 69 (25%) infected ducks developed a variety of neoplasms, including lymphosarcomas, histiocytic sarcomas, and spindle-cell sarcomas.

Cytopathic effects and focus formation by reticuloendotheliosis viruses in a quail fibroblast cell line.

The T and CS (chick syncytial) strains of reticuloendotheliosis virus (REV) induced focus formation and cytonecrotic changes in a Japanese quail fibroblast cell line designated QT35. The focus was distinctive and readily discernible, consisting of rounded refractile and larger dark-appearing (syncytia) cells usually aggregated along the periphery of a hole within a focus. The foci and cytopathic effects induced by the two strains of REV were morphologically indistinguishable. The focus formation in QT35 cultures was inhibited by chicken antiserum against the homologous or heterologous strain of REV; the homologous antiserum induced a greater inhibition. The altered cells composed of focus exhibited cytoplasmic fluorescence when stained by an indirect fluorescent-antibody technique with either the homologous or heterologous anti-REV serum. When foci that developed in QT35 cultures maintained under fluid medium were enumerated 3 days postinoculation, there were a highly significant correlation (P less than 0.001) and a linear relationship between the number of foci developed and relative virus concentration inoculated, thus justifying the validity of focus assay of REV by this system. The QT35 cell line may provide a useful in vitro system for virological and serological studies of REV.

An IgM-producing B lymphoblastoid cell line established from lymphomas induced by a non-defective reticuloendotheliosis virus.

Chick syncytial virus (CSV), a strain of avian reticuloendotheliosis virus (REV) causes lymphoid tumours in chickens after a prolonged incubation period. A number of CSV-induced tumours were examined for cell surface antigen and were found to be of the B cell type and to produce immunoglobulin. Attempts were made to grow in vitro cell lines from CSV-induced tumours and a lymphoblastoid cell line was established from a liver tumour of a chicken that was inoculated with CSV via the yolk sac in embryo. The donor chicken was viraemic at the time the tumour was removed. The cell line is designated RECC-RP13, it produces non-defective REV, is a B cell type and it produces IgM. It is free from infection with endogenous and exogenous avian leukosis virus (ALV) and has an increased number of chromosomes. Sequences specific to REV were detected in at least four sites in cellular DNA from RECC-RP13. Sequences specific to ALV DNA, beyond that normally found in 151(5) X 7(1) cells, were not found in DNA from this cell line.

Cell killing by spleen necrosis virus is correlated with a transient accumulation of spleen necrosis virus DNA.

Spleen necrosis virus productively infects avian and rat cells. The average number of molecules of unintegrated and integrated viral DNA in cells at different times after infection was determined by hybridization and transfection assays. Shortly after infection, there was a transient accumulation of an average of about 150 to 200 molecules of unintegrated linear spleen necrosis virus DNA per chicken, turkey, or pheasant cell. No such accumulation was seen in infected rat cells. Soon after infection there was in chicken cells, but not inturkey, pheasant, or rat cells, also a transient integration of an average of 35 copies of viral DNA per cell. By 10 days after infection, the majority of this integrated viral DNA was lost from the population of infected chicken cells. At the same time, the majority of the unintegrated viral DNA was also lost from infected chicken, turkey, and pheasant cells. The transient cytopathic effect seen in these infected cells also occurred at this time. Late after infection about five copies of apparently nondefective spleen necrosis proviruses were stably integrated at multiple sites in chicken, turkey, pheasant, and rat DNA. These results demonstrate a correlation between the transient accumulation of large numbers of spleen necrosis virus DNA molecules and the transient occurrence of cytopathic effects.

Complement-fixation test for reticuloendotheliosis viruses: limits of sensitivity in infected avian cells.

A specific micro-complement-fixation procedure for assay of avian reticuloendotheliosis viruses (REV), designated by use as the COFAR test, was compared with an assay based on immunofluorescent (IF) antibody staining of infected chick embryo fibroblasts. Endpoint titrations in which REV strain T, chick syncytial virus, and spleen necrosis virus were used indicated that cultures infected with limiting dilutions of each strain were positive by both procedures within 6 days. Depending on cell density, infection of 2 to 9% of the cells cultured produced an unambiguous positive response (titer greater than or equal to 1:2) with COFAR test. When both tests were used in a study on the transmission and in vivo status of ducks infected with spleen necrosis virus, COFAR was no less, possibly more, sensitive than IF for detecting infection in cultures inoculated with plasma.

Formation of reticuloendotheliosis virus pseudotypes of Rous sarcoma virus.

Superinfection of chicken embryo fibroblasts transformed by the defective Bryan strain of Rous sarcoma virus (BH-RSV) with two different reticuloendotheliosis viruses (REVs), REV strain T (REV-T) or spleen necrosis virus (SNV), resulted in the production of infectious sarcoma virus pseudotypes. These pseudotypes were neutralized by antiserum prepared against SNV and were unable to infect chicken cells preinfected with either REV-T or SNV. These results suggest that defective BH-RSV is able to use the glycoprotein from REV to form infectious pseudotypes. On the other hand, neither REV-T nor SNV was able to supply a functional reverse transcriptase to the polymerase-negative mutant BH-RSValpha, nor was REV-T or SNV able to complement the defect in the internal protein gene of the temperature-sensitive avian sarcoma virus mutant NY45.

Characteristics of two new reticuloendotheliosis virus isolates of turkeys.

Reticuloendotheliosis (RE) virus strains MN81 and MN67 isolated from epiornithics of RE in turkeys were partially characterized. Strains MN81 and MN67 replicated in chicken embryo fibroblast,duck embryo fibroblast and turkey embryo-fibroblast cultures and produced syncytial cytopathic effects in duck embryo fibroblast and turkey embryo fibroblast cultures. The virions of MN81 and MN67 measured approximately 100 nm in diameter, resembled RE virus strain T, and could be distinguished from avian leukosis viruses morphologically. The buoyant density of strain MN81 was found to be 1.15 g/cm3 in sucrose gradients. Strains MN81 and MN67 were inactivated by heat, acid pH, ether, and chloroform treatments. These strains were serologically unrelated to avian leukosis virus but were related to RE virus strains T, CS, DIA, and SN.